The plasma membrane of a cell separates the cytoplasm of the cell from the environment, and it is primarily composed of a phospholipid bilayer and proteins embedded within the bilayer or attached to the surface thereof. Normally the plasma membrane functions as a gatekeeper, which controls trafficking of essential substances into and out of the cell. However, the cell plasma membrane also functions as permeability barrier and blocks the passage of many useful therapeutic agents. Generally, hydrophilic molecules, highly charged molecules and macromolecules such as nucleic acid or gene cannot readily cross the cell membranes. Therefore, there is a need for a reliable means of transporting drugs and macromolecules into the cell.
Heretofore, a number of transporter molecules such as lipids, polymers and dendrimers have been proposed to escort molecules across biological membranes, but often they are not easily water soluble or biodegradable, and hence, precipitate in a cell to illicit toxicity.
Proteins having a PTD (protein transduction domain) that allows the protein permeation through the plasma membrane include the HIV-1 Tat peptide, Antennapedia (Antp) homeodomain protein, Herpes virus protein VP22, Nuclear localization signal (NLS) sequence, and the like, as shown in Table 1.
TABLE 1ProteinSEQhaving PTDAmino acid sequenceID NO:HIV-1 TatGRKKRRQRRRPPQ1(48-60 a.a.) Antp (43-58 a.a.)RQIKIWFQNRRMKWKK2 VP22DAATATRGRSAASRPTERDRAPA3(267-300 a.a.)RSASRPRRPVE SV40-NLSPKKKRKVC4 NucleoplasminKRPAAIKKAGQAKKKKC5 NF-κBPMLKQRKRQA6 HIV-1 RevRQARRNRRRRWRERQRG7(34-50 a.a.) FHV CoatRRRNRTRRNRRRVRRGC8(35-49 a.a.)
The above protein domains seem to facilitate the permeation across biological membranes without the help of any specific transporter or receptor associated with the cell. Further, they contain a high percentage of basic amino acids such as arginine and lysine. The basic region (i.e., 49-57 a.a.) of the Tat protein, which is a necessary transacting transcriptional activator of HIV virus reproduction, has been reported to play a critical role in the process of the protein permeation through the plasma membrane. A number of studies have reported syntheses of various oligopeptides having a multiple units of arginine and used them as molecular transporters.
From these studies, it has been found that oligomers having eight to nine arginine residues show the highest permeability and are most effective in enhancing the transportation of molecules attached thereto across a biological membrane, which suggests that the guanidine group of arginine plays an essential role in the transportation of molecules attached thereto across a biological membrane.
Wender et al. designed peptoid molecular transporters based on the fact that the biological membrane permeability of a peptide largely depends on the number of the guanidine group in the peptide, the length of the linker chain, and chirality, etc. It was found that an L-arginine nonamer is 20-times more effective in the transportation across a biological membrane than Tat protein (49-57 a.a.), and D-arginine nonamer was also significantly more effective in the uptake by Jurkat cells, as was determined using FACS (P. A. Wender, et al., Proc. Natl. Acad. Sci. U.S.A. 97: 13003, 2000). These results suggest that the permeability of peptides having a certain number of guanidine groups is not significantly affected by the chirality of the amino acid (U.S. Pat. No. 6,495,663; Korean Patent Laid-Open Publication No. 2001-12809).
However, such polyarginine peptide or related peptoid molecules have problems of rapid metabolism to be eliminated through the liver and kidney as well as their in vivo toxicity liability. Further, the fact that a peptide or peptoid having a number of guanidine groups can maintain its helical structure only in a basic environment suggests that its membrane permeability depends largely on the positively charged guanidinium groups rather then the secondary or tertiary structure thereof.
The present inventors have therefore endeavored to develop molecular transporters prepared by introducing positively charged guanidinium groups to sugar or its analogue structure in a linear or branched form with a high density of functionality and found that such molecular transporters significantly enhance the transport of various physiologically active molecules attached thereto either covalently or ionically, across a biological membrane.